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ABSTRACT In this study, the microbial community in the anaerobic/oxic/anoxic (A/O/A) process combined with sludge ozonation and phosphorus recovery was characterized by using phosphorus uptake rate (PUR) analysis and PCR-cloning analysis. Despite effective phosphorus removal, PUR analysis indicated a lower activity of both polyphosphate-accumulating organisms (PAOs) and denitrifying PAOs (DNPAOs) than in other systems utilizing DNPAOs. This result suggested that endogenous denitrifying bacteria actively contributed to denitrification. The PCR-cloning analysis revealed that Bacteroidetes was most prominent in the process, followed by Betaproteobacteria and Alphaproteobacteria. For Bacteroidetes, most of the sequences obtained in this study were not closely related to isolates. On the other hand, for the Alphaproteobacteria, the genera Amaricoccus, Aminobacter, Hyphomicrobium, and Paracoccus, which have the ability both to accumulate poly-β-hydroxybutyrate (PHB) and to reduce nitrate to nitrite, were detected. For the Betaproteobacteria, which are major denitrifying bacteria in wastewater treatment systems, the genera Dechloromonas and Zoogloea, were identified. Organisms belonging to the family Comamonadaceae, some of which have been reported as being primary poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)-degrading denitrifying bacteria, also existed in the system. Major PAOs/DNPAOs, Rhodocyclus-related PAOs and Actinobacterial PAOs, were not detected, suggesting that unknown PAOs/DNPAOs could have played an important role for phosphorus removal
Journal of Water and Environment Technology, Vol. 7, No. 3, 2009 - 155 - Characterization of the microbial community in the anaerobic/oxic/anoxic process combined with sludge ozonation and phosphorus adsorption Takashi KONDO*, Satoshi TSUNEDA**, Yoshitaka EBIE*, Yuhei INAMORI***, and Kaiqin XU* * Research Center for Material Cycles and Waste Management, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan ** Department of Life Science and Medical Bioscience, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan *** Faculty of Symbiotic Systems Science, Fukushima University, 1 Kanayagawa, Fukushima, Fukushima 960-1296, Japan ABSTRACT In this study, the microbial community in the anaerobic/oxic/anoxic (A/O/A) process combined with sludge ozonation and phosphorus recovery was characterized by using phosphorus uptake rate (PUR) analysis and PCR-cloning analysis. Despite effective phosphorus removal, PUR analysis indicated a lower activity of both polyphosphate-accumulating organisms (PAOs) and denitrifying PAOs (DNPAOs) than in other systems utilizing DNPAOs. This result suggested that endogenous denitrifying bacteria actively contributed to denitrification. The PCR-cloning analysis revealed that Bacteroidetes was most prominent in the process, followed by Betaproteobacteria and Alphaproteobacteria. For Bacteroidetes, most of the sequences obtained in this study were not closely related to isolates. On the other hand, for the Alphaproteobacteria, the genera Amaricoccus, Aminobacter, Hyphomicrobium, and Paracoccus, which have the ability both to accumulate poly-β-hydroxybutyrate (PHB) and to reduce nitrate to nitrite, were detected. For the Betaproteobacteria, which are major denitrifying bacteria in wastewater treatment systems, the genera Dechloromonas and Zoogloea, were identified. Organisms belonging to the family Comamonadaceae, some of which have been reported as being primary poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)-degrading denitrifying bacteria, also existed in the system. Major PAOs/DNPAOs, Rhodocyclus-related PAOs and Actinobacterial PAOs, were not detected, suggesting that unknown PAOs/DNPAOs could have played an important role for phosphorus removal. Keywords: Denitrifying PAOs (DNPAOs), microbial community analysis, phosphorus uptake rate (PUR) INTRODUCTION Activated sludge processes have been widely used to effectively remove nutrients from municipal/industrial wastewater. In wastewater treatment plants (WWTPs), treatment and disposal of excess sludge have been serious problems, and the treatment of excess sludge may account for as much as 25% to 65% of total plant operating costs (Liu, 2003). Furthermore, the regulations regarding disposal have become increasingly strict in most countries (Liu, 2003; Ødegaard, 2004). In related work, to achieve effective sludge reduction and phosphorus recovery, a lab- scale continuous anaerobic/oxic/anoxic (A/O/A) process equipped with an ozonation system and a phosphorus adsorption column used in a previous study was improved (Kondo et al., 2009). In the improved process, excess sludge was solubilized by microbubble ozonation. Then, the supernatant after ozonation was passed to the Address correspondence to Satoshi Tsuneda, Department of Life Science and Medical Bioscience, Waseda University, Email: stsuneda@waseda.jp Received 1 February 2009, Accepted 18 May 2009 Journal of Water and Environment Technology, Vol. 7, No. 3, 2009 - 156 - phosphorus adsorption column, which was packed with zirconium-ferrite (ZrFe 2 (OH) 8 ) adsorbent. The supernatant was circulated back to the anaerobic and oxic tanks. The solid materials after both ozonation and phosphorus adsorption were added to the anaerobic tank (Fig. 1). In operation with rural wastewater, effective nutrient removal and phosphorus recovery were achieved with no excess sludge production for at least 2 months (Kondo et al., 2009). In the process, phosphorus was accumulated in the biomass by polyphosphate-accumulating organisms (PAOs) in the oxic tank and denitrifying PAOs (DNPAOs) in the anoxic tank. For the nitrogen removal, denitrification occurred in the anoxic tank without organic carbon sources, suggesting that endogenous denitrifying bacteria including DNPAOs reduced nitrate/nitrite with the use of an intercellular energy source (Kondo et al., 2009). In this study, to estimate both the proportion of DNPAOs to total PAOs and the proportion of DNPAOs to total endogenous denitrifying bacteria, phosphorus uptake ratio (PUR) analysis was conducted. Then, PCR-cloning analysis was performed to identify organisms playing important roles for nutrient removal in the process. Fig. 1 - Schematic Diagram of the A/O/A Process with Ozonation and Phosphorus Adsorption (Kondo et al., 2009). MATERIALS AND METHODS Reactor operation and sampling Sludge samples for the PUR analysis and the PCR-cloning analysis were collected from the lab-scale continuous A/O/A process equipped with the ozonation system and phosphorus adsorption column which was operated in the related study (Kondo et al., 2009). The working volume was 36 L (anaerobic tank, 10.3 L; oxic tank, 10.3 L; anoxic tank, 15.4 L). The ozonation system, with a working volume of 20 L, received excess sludge from the anoxic tank, and the amount of sludge was adjusted to 9.4 % of total MLSS per day. Ozonation was performed twice a week, and then the supernatant of the ozonated sludge was flowed into the phosphorus adsorption column which was filled with 1.5 L of spherical zirconium-ferrite (ZrFe 2 (OH) 8 ) adsorbent (Japan Enviro Chemicals, Japan). Sixty percent of the mixture of effluent and backwash water from the column was circulated back to the anaerobic tank, and the residue was circulated back to the oxic tank. The solid residuals after ozonation were added to the anaerobic tank. The characteristics of the influent wastewater were as follows: 160–200 mg/L of SS, 55–80 mg/L of TOC, 45–55 mg/L of T-N, and 4.0–5.5 mg/L of T-P. Other details are shown in the related study (Kondo et al., 2009). Sampling was performed once efficient Journal of Water and Environment Technology, Vol. 7, No. 3, 2009 - 157 - nutrient removal efficiency was maintained for more than one month (TOC <10 mg/L; T-N <10 mg/L; T-P <1 mg/L). Phosphorus uptake rate (PUR) analysis The phosphorus uptake rate (PUR) was determined according to the method described in the literature, with some modification (Kuba et al., 1997; Tsuneda et al., 2006; Wachtmeister et al., 1997). In brief, 200 mL of activated sludge was collected from the settling tank and transferred to a 500 mL polystyrene cup. The same volume of influent raw rural wastewater was added to the cup and incubated for 90 min anaerobically. After anaerobic incubation, the sludge sample was divided into two cups. One was exposed to oxic conditions, and the other was exposed to anoxic conditions (use of nitrogen gas and addition of 20 mg-N/L NaNO 3 to the cup). The PUR was estimated from the slope of the line describing the linear decrease in phosphate concentration. The ratio of anoxic PUR to aerobic PUR (anoxic/oxic PUR ratio) was used as an index reflecting the fraction of DNPAOs (Kuba et al., 1997; Tsuneda et al., 2006; Wachtmeister et al., 1997). The ratio of anoxic PUR to denitrification rate under anoxic conditions was also determined (Kuba et al., 1997). Analytical methods MLSS was measured according to the procedure described in Standard Methods (1995). To determine soluble TOC (S-TOC), NH 4 -N, NO 2+3 -N, NO 2 -N, and PO 4 -P, water samples were filtered using a glass-fiber filter (GF/C, Whatman Japan KK, Japan). TOC and S-TOC were measured using a SHIMADZU TOC-VSCH (Shimadzu, Japan). Total nitrogen (T-N), total phosphorus (T-P), soluble T-N (ST-N), soluble T-P (ST-P), NH 4 -N, NO 2+3 -N, NO 2 -N, and PO 4 -P were measured using a TRAACS 2000 (Bran+Luebbe, Japan). Microbial community analysis using PCR-Cloning For PCR-cloning analysis, a sludge sample was collected and subjected to total DNA extraction using Isoplant (Nippon Gene, Japan). Then, the 16S rRNA genes were amplified by PCR amplification using universal primers, 341f and 907r (Muyzer et al., 1998). PCR product purification, cloning, plasmid DNA preparation, and sequencing with an ABI PRISM 3100-Avant DNA Sequencing system (Applied Biosystems, Japan) were performed as described previously (Osaka et al., 2006). A database search was conducted using BLAST from the DDBJ. The sequences determined in this study and those retrieved from the database were aligned using Clustal W (Thompson et al., 1994). Phylogenetic trees were constructed using Clustal W and Tree View (Page, 1996) using the neighbor-joining method (Saitou and Nei, 1987). RESULTS AND DISCUSSION PUR analysis To confirm the proportion of the phosphorus accumulation activity of DNPAOs to that of total PAOs, the PUR was estimated and compared with other systems in which the contribution of DNPAOs to phosphorus removal is known (Table 1). In this study, both the oxic and anoxic PURs were lower than those in other systems, indicating the low activity of PAOs in the process. In the University of Cape Town (UCT) processes, the PURs were slightly high but were different among processes (Kuba et al., 1997). Kuba Journal of Water and Environment Technology, Vol. 7, No. 3, 2009 - 158 - et al. (1997) explained that the low DNPAO activity was due to (i) nitrate/oxygen transfer into the anaerobic/anoxic tanks, (ii) the low retention time of wastewater in the sewer line (lower composition of fatty acids), and (iii) low nitrogen loading to the anoxic tank. In this study, the nitrate concentration in the influent to the anoxic tank was around 20 mg-N/L (Kondo et al., 2009), which was not lower than those in the UCT processes (Kuba et al., 1997). On the other hand, oxygen transfer must have occurred because the anoxic tank follows the oxic tank, whereas less nitrate transfer might occur in the anaerobic tank (both the influent wastewater and return sludge (determined as effluent) contained less than 0.5 mg/L of nitrate). The composition of fatty acids also must be low. The wastewater used in this study was collected from a rural wastewater treatment facility where the retention time of wastewater in the sewer line is lower than those in the UCT processes. In sequencing batch reactors (SBRs), the PURs are higher than the others (Table 1). These high PURs were due not to the differences in the process types but to the differences in influent wastewater: these SBRs received synthetic wastewater containing acetate as the main organic carbon to enrich the PAO/DNPAO population. Table 1 Summary of kinetics in the PUR analysis of this study and previous studies. The proportion of anoxic PUR to oxic PUR (anoxic PUR/oxic PUR) was almost the same as the UCT processes and one A/O/A SBR (Kuba et al., 1997; Tsuneda et al., 2006). Anoxic PUR/oxic PUR in the anaerobic/anoxic (A/A) SBR, in which DNPAOs dominated, was much higher than that in this study. These results demonstrated that the proportion of DNPAO activity for phosphorus accumulation to total PAO activity in this study was almost the same as other processes utilizing DNPAOs, whereas both DNPAOs and oxygen-utilizing PAOs contributed to phosphorus removal. In the A/O/A process, nitrite/nitrate was reduced to nitrogen in the anoxic tank. The ratios of anoxic PUR to denitrification rate (P/N ratio) in this study and previous studies are shown in Table 1. The P/N ratio in this study was substantially lower than that in previous studies. This suggests that other denitrifying bacteria that had no ability to accumulate phosphorus contributed to denitrification in the A/O/A process. Microbial community in the A/O/A process In PCR-cloning analysis, a total of 142 clones were amplified and classified as 98 operation taxonomical units (OTUs). All OTUs comprised less than 6 clones, suggesting that the organisms in the A/O/A process with sludge ozonation and phosphorus recovery belonged to very different phylogenetic groups, and there was no prominent species. Journal of Water and Environment Technology, Vol. 7, No. 3, 2009 - 159 - The numbers of clones affiliated with different divisions and subdivisions are shown in Table 2. Most clone sequences were affiliated with the Bacteroidetes (33 clones belonging 23 OTUs), followed by the Betaproteobacteria (30 clones belonging to 25 OTUs) and the Alphaproteobacteria (22 clones belonging to 16 OTUs). Most sequences were closely related to the sequences obtained from activated sludge in previous studies (Hoshino et al., 2006; Osaka et al., 2006). Table 2 Phylogenetic affiliations and numbers of 16S rRNA gene clones. For the Bacteroidetes, except for the relative sequences (AOA-clone 37 and 106) to the genus Runella, no isolate was closely related to the sequences obtained in this study (Fig. 2A). Moreover, most of the sequences belonging to the Bacteroidetes were not closely related to any sequences in the database (below 97% similarity). The genus Runella isolated from diverse environmental habitats and Runella limosa, which has no ability to accumulate phosphorus and reduce nitrate to nitrite, was recently isolated from an acetate-fed SBR with efficient phosphorus removal (Ryu et al., 2006). For the Alphaproteobacteria (Fig. 2B), most of the sequences were affiliated with the isolated bacteria, the genera Amaricoccus, Aminobacter, Beijerinckia, Deyosia, Hyphomicrobium, Nordella, Paracoccus and Roseomonas. Maszenan et al. (1997) reported that the genus Amaricoccus isolated from activated sludge has been capable of both poly-β-hydroxybutyrate (PHB) accumulation in oxic conditions and nitrate reduction to nitrate. According to Bergey’s Manual of Determinative Bacteriology (1994), the genus Beijerinckia is not denitrifying bacteria but PHB accumulating bacteria, and the genera Aminobacter, Hyphomicrobium, and Paracoccus have the ability to both accumulate PHB and to reduce nitrate to nitrite. For the Betaproteobacteria (Fig. 2C), 8 OTUs (11 clones) were affiliated with the family Comamonadaceae. Recently, the family Comamonadaceae has been reported as being a primary poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)-degrading denitrifying bacteria (Khan et al., 2002, 2007). These bacteria could utilize the solid PHBV as an electron donor under denitrifying conditions (Khan et al., 2002). The genera Dechloromonas and Zoogloea, which are major denitrifying bacteria, were identified in this study, and these denitrifying bacteria might utilize these intermediates. One of the major PAOs/DNPAOs, Rhodocyclus-related PAOs (RPAOs, Candidatus “Accumulibacter phosphatis”), was not detected in the process. Journal of Water and Environment Technology, Vol. 7, No. 3, 2009 - 160 - (A) (B) (C) Fig. 2 - Phylogenetic dendrogram based on 16S rRNA gene sequences in relation to members of the Bacteroidetes (A), Betaproteobacteria (B), and Alphaproteobacteria (C). The root of the tree was determined by using the 16S rRNA gene of Methanosarcina mazei (AB065295) as the out group. Scale bar indicates the 10% estimated difference in nucleotide sequence position. Clones obtained in this study are shown in boldface and labeled ‘AOA-clone’. The number of clones obtained is shown in parentheses. Journal of Water and Environment Technology, Vol. 7, No. 3, 2009 - 161 - Considering the results of the PCR-cloning analysis, some denitrifying organisms that do not accumulate polyphosphates were identified. Intercellular PHB accumulating Alphaproteobacteria such as the genus Hyphomicrobium accumulated PHB in the anaerobic or oxic tanks, and PHB was utilized as an energy source for endogenous denitrification in the anoxic tank. The genera Dechloromonas and Zoogloea, major denitrifying bacteria belonging to Betaproteobacteria, were detected in the system. The contribution of these denitrifying bacteria resulted in the low P/N ratio in the PUR analysis. Organisms belonging to the family Comamonadaceae might also have contributed to denitrification in the system. Major PAOs, Rhodocyclus-related PAOs and Actionobacterial PAOs, were not detected in the PCR analysis, suggesting that unknown PAOs play an important role for phosphorus removal in the system. CONCLUSIONS In this study, PUR analysis and PCR-cloning analysis were performed to characterize the microbial community in the A/O/A process with sludge ozonation and phosphorus recovery. The main conclusions are as follows. (1) PUR analysis indicated a lower activity for phosphorus accumulation of both PAOs and DNPAOs than other processes employing DNPAOs, whereas the proportion of DNPAO activity was almost the same. The lower P/N ratio than other processes suggested that other denitrifying bacteria contributed to denitrification. (2) The organisms in the process belonged to very different phylogenetic groups, and there was no prominent species. 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